The field of this invention is proteins involved in chromatin destabilization.
Apoptosis is executed through a suicide program that is built into all animal cells (reviewed by White,1996; Wyllie,1995). Cells undergoing apoptosis show distinctive morphological changes, including membrane blebbing, cytoplasmic and nuclear condensation, chromation aggregation and formation of apoptotic bodies (Wyllie, 1980). The biochemical hallmark of apoptosis is the cleavage of chromatin into nucleosomal fragments (Wyllie et al., 1980). Multiple lines of evidence indicate that apoptosis can be triggered by the activation of a family of cysteine proteases with specificity for aspartic acid residues, including CED-3 of C. elegans, CPP32/Yama/Apopain of humans, and DCP-1 of Drosophila (Yuan et al., 1993; Xue et al., 1996; Fernandes-Alnemri, et al., 1994; Tewari, et al., 1995; Nicholson, et al., 1995; Song, et al., 1997). Recently, these proteins have been designated as caspases (Alnemri et al., 1996).
The most intensively studied apoptotic caspase is caspase-3, previously called CPP32/Yama/Apopain (Fernandes-Alnemri, et al., 1994; Tewari, et al., 1995; Nicholson, et al., 1995). Caspase-3 normally exists in the cytosolic fraction of cells as an inactive precursor that is activated proteolytically when cells are signaled to undergo apoptosis (Schlegel et al., 1996; Wang et al., 1996). Multiple apoptotic signals, including serum withdrawal, activation of Fas, treatment with granzyme B, ionizing radiation, and a variety of pharmacological agents, activate caspase-3 (Chinnaiyan et al., 1996; Darmon, et al., 1996; Datta et al., 1996, 1997; Erhardt and Cooper, 1996; Hasegawa et al., 1996; Jacobson et al., 1996; Martin et al., 1996; Schlegel et al, 1996). A caspase-3-specific tetrapeptide inhibitor, Ac-DEVD SEQ ID NO:3-CHO, can abolish the ability of cytosol from apoptotic cells to induce apoptosis in normal nuclei and block the initiation of the cellular apoptotic program in response to apoptotic stimuli (Nicholson et al., 1995; Dubrez, et al., 1996; Jacobson et al., 1996). Deletion of caspase-3 from the mouse genome through homologous recombination results in excessive accumulation of neuronal cells, owing to a lack of apoptosis in the brain (Kuida et al., 1996). Addition of active caspase-3 to normal cytosol activates the apoptotic program (Enari et al., 1996). These data indicate that caspase-3 is both necessary and sufficient to trigger apoptosis.
The identified substrates of caspase-3 include poly(ADP-ribose) polymerase (PARP) (Tewari et al. 1995; Nicholson et al., 1995), sterol-regulatory element binding proteins (SREBPs) (Wang et al., 1995; 1996), the U1 associated 70 kDa protein (Caciola-Rosen et al. 1996), D4-GDI (Na et al., 1996), huntingtin (Goldberg et al., 1996), and the DNA-dependent protein kinase (Casciola-Rosen et al., 1996; Song et al., 1996). It is not known whether the cleavage of any of these substrates plays a causal role in apoptosis.
Our laboratory recently established an experimental system in which DNA fragmentation characteristic of apoptosis can be triggered in vitro by incubation of normal nuclei with activated cytosolic extracts (Liu et al., 1996b). The activation occurred in two stages: first, cytosolic caspase-3 was cleaved and activated in a reaction that was triggered by cytochrome c released from mitochondria; and second, activated caspase-3 interacted with other cytosolic components to generate DNA fragmentation when added to isolated nuclei (Liu et al., 1996b; Yang et al., 1997). The present invention describes the purification, characterization, and cDNA cloning of a downstream factor that is activated by caspase-3 and in turn induces nuclear DNA fragmentation. We call this factor DNA Fragmentation Factor (DFF).
The invention provides methods and compositions relating to isolated DNA Fragmentation Factor (DFF) polypeptides, related nucleic acids, and polypeptide domains thereof having DFF-specific activity. DFF polypeptides can regulate chromatin stability and hence provide important regulators of cell viability. The polypeptides may be produced recombinantly from transformed host cells from the subject DFF encoding nucleic acids or purified from mammalian cells. The invention provides isolated DFF hybridization probes and primers capable of specifically hybridizing with the disclosed DFF gene, DFF-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for DFF transcripts), therapy (e.g. gene therapy to modulate DFF gene expression) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating other transcriptional regulators, reagents for screening chemical libraries for lead pharmacological agents, etc.).
The DNA Fragmentation Factor (DFF) polypeptides of the invention include DFF-45, DFF-40, and polypeptides comprising domains thereof. Such DFF domains have at least 10, preferably at least about 12, more preferably at least about 14 consecutive residues of DFF-45 or DFF-40 and provide DFF domain specific activity or function, such as being a caspase-3 substrate, mediating DNA fragmentation, activating a nuclease and inhibiting a DFF polypeptide.
The nucleotide sequence of a natural cDNA encoding a human DFF-45 polypeptide is shown as SEQ ID NO:1, and the full conceptual translate is shown as SEQ ID NO:2. The DFF polypeptides of the invention include incomplete translates of SEQ ID NO:1 and deletion mutants of SEQ ID NO:2, which translates and deletion mutants have DFF-specific amino acid sequence and binding specificity or function.
DFF-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of an DFF polypeptide with a binding target is evaluated. The binding target may be a natural intracellular binding target such as another DFF polypeptide, a DFF regulating protein, a DFF-activated nuclease, or other regulator that directly modulates DFF activity or its localization; or non-natural binding target such a specific immune protein such as an antibody, or an DFF specific agent such as those identified in screening assays such as described below. DFF-binding specificity may assayed by binding equilibrium constants (usually at least about 107 Mxe2x88x921, preferably at least about 108 Mxe2x88x921, more preferably at least about 109 Mxe2x88x921), by the ability of the subject polypeptide to function as negative mutants in DFF-expressing cells, to elicit DFF specific antibody in a heterologous host (e.g a rodent or rabbit), etc. In any event, the DFF binding specificity of the subject DFF polypeptides necessarily distinguishes AIF.
The claimed DFF polypeptides are isolated or pure: an xe2x80x9cisolatedxe2x80x9d polypeptide is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total polypeptide in a given sample and a pure polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample. The DFF polypeptides and polypeptide domains may be synthesized, produced by recombinant technology, or purified from mammalian, preferably human cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art.
The invention provides natural and non-natural DFF-specific binding agents, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, in addition to functions disclosed below, chromatin stability may be dependent on the stability of certain DFF complexes and is subject to regulation by modulating the stability of the DFF complex. Hence, agents which modulate the stability of DFF complexes such as DFF-45/DFF-40 provide means for regulating cell growth and viability. Such agents have the advantage of bypassing many mechanisms of MDR frequently associated with neoproliferative disease. Novel DFF-specific binding agents include DFF-specific receptors, such as somatically recombined polypeptide receptors like specific antibodies or T-cell antigen receptors (see, e.g Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural intracellular binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries such as described below, etc. For diagnostic uses, the binding agents are frequently labeled, such as with fluorescent, radioactive, chemiluminescent, or other easily detectable molecules, either conjugated directly to the binding agent or conjugated to a probe specific for the binding agent. Agents of particular interest modulate DFF function, e.g. DFF-dependent apoptosis; for example, isolated cells, whole tissues, or individuals may be treated with an DFF binding agent to activate, inhibit, or alter DFF-dependent apoptotic processes.
The amino acid sequences of the disclosed DFF polypeptides are used to back-translate DFF polypeptide-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et. al. (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural DFF-encoding nucleic acid sequences (xe2x80x9cGCGxe2x80x9d software, Genetics Computer Group, Inc, Madison Wis.). DFF-encoding nucleic acids used in DFF-expression vectors and incorporated into recombinant host cells, e.g. for expression and screening, transgenic animals, e.g. for functional studies such as the efficacy of candidate drugs for disease associated with DFF-modulated cell viability, etc.
The invention also provides nucleic acid hybridization probes and replication/amplification primers having a DFF cDNA specific sequence contained in SEQ ID NO:1 and sufficient to effect specific hybridization thereto (i.e. specifically hybridize with SEQ ID NO:1 in the presence of HeLa cell cDNA). Such primers or probes are at least 12, preferably at least 24, more preferably at least 36 and most preferably at least 96 bases in length. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5xc3x97SSPE (0.18 M NaCl, 0.01 M NaPO4, pH7.7, 0.001 M EDTA) buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5xc3x97SSPE buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE buffer at 42xc2x0 C. DFF cDNA homologs can also be distinguished from other polypeptide using alignment algorithms, such as BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410).
The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Nucleic acids comprising the nucleotide sequence of SEQ ID NO:1 or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.
The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of DFF genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional DFF homologs and structural analogs. In diagnosis, DFF hybridization probes find use in identifying wild-type and mutant DFF alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic DFF nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active DFF.
The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of a DFF modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate DFF interaction with a natural DFF binding target. A wide variety of assays for binding agents are provided including labeled in vitro protein-protein binding assays, immunoassays, cell based assays, etc. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
In vitro binding assays employ a mixture of components including an DFF polypeptide, which may be part of a fusion product with another peptide or polypeptide, e.g. a tag for detection or anchoring, etc. The assay mixtures comprise a natural intracellular DFF binding target. While native binding targets may be used, it is frequently preferred to use portions (e.g. peptides) thereof so long as the portion provides binding affinity and avidity to the subject DFF polypeptide conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.
The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the DFF polypeptide specifically binds the cellular binding target, portion or analog with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening.
After incubation, the agent-biased binding between the DFF polypeptide and one or more binding targets is detected by any convenient way. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. Separation may be effected by precipitation (e.g. TCA precipitation, immunoprecipitation, etc.), immobilization (e.g on a solid substrate), etc., followed by washing by, for examples, membrane filtration, gel chromatography (e.g. gel filtration, affinity, etc.). For cell-based DFF-dependent DNA fragmentation, xe2x80x98bindingxe2x80x99 is detected by a change in the chromatin stability of the cell.
Detection may be effected in any convenient way. For cell-free binding assays, one of the components usually comprises or is coupled to a label. The label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, an enzyme, etc. A variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g. through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc.
A difference in the binding affinity of the DFF polypeptide to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the DFF polypeptide to the DFF binding target. Analogously, in the cell-based assay also described below, a difference in the DFF-dependent chromatin degredation or instability in the presence and absence of an agent indicates the agent modulates DFF function. A difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.
The following experimental section and examples are offered by way of illustration and not by way of limitation.